PDF(Pubmed)
Abstract：
Up to a third of the world\'s population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.
摘要：
世界上三分之一的人口患有过敏症,然而，现有预防措施的有效性仍然存在，总的来说，穷。因此,开发成功的预防策略来诱导对过敏原的耐受是至关重要的.在概念验证研究中，我们的实验室先前已经表明，自体造血干细胞（HSC）或表达主要草花粉过敏原的自体B细胞的转移，Phlp5在小鼠中诱导强烈的耐受性。然而,最终的临床翻译将需要安全的过敏原表达而不需要逆转录病毒转导。因此,我们旨在将Phlp5化学偶联到白细胞表面，并测试其诱导耐受的能力。Phlp5通过两种独立的技术耦合，通过1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）或通过亲脂性锚连接，1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-聚(乙二醇)-马来酰亚胺(DSPE-PEG-Mal)。通过流式细胞术在新鲜和培养的Phlp5偶联细胞中评估有效性，图像细胞计数,和免疫荧光显微镜。使用EDC的Phlp5的化学偶联是稳健的,但随后是快速凋亡。DSPE-PEG-Mal介导的连锁也很强，但由于抗原内化，抗原水平下降。将通过任一种方法与Phlp5偶联的细胞转移到自体小鼠中。虽然EDC偶联脾细胞与短期免疫抑制一起施用最初将Phlp5特异性抗体水平降低到中等程度，两种方法在多次皮下免疫过敏原后均未诱导对Phlp5的持续耐受.总的来说,我们的结果表明，使用两种独立的技术成功地将过敏原与白细胞进行化学连接，消除遗传修饰的风险。仍然需要实现更持久的表面表达以用于预防性细胞治疗方案。
